P452: CD4+ T CELLS REDUCE THE POPULATION OF LEUKEMIC STEM CELLS IN ACUTE MYELOID LEUKEMIA
Authors: Noah Schnüriger, Walid Gharib, Irene Keller, Lea Taylor, Remy Bruggmann, Adrian Ochsenbein, Carsten Riether
Journal: HemaSphere (2023)

Background: Acute myeloid leukemia (AML) is a very heterogeneous, hematologic disorder. Although, its pathogenesis is understood well, it remains challenging to treat. This malignant disease originates from and is maintained by leukemic stem cells (LSCs) which reside in the bone marrow (BM). A major obstacle for successful therapy is the efficient and sustainable eradication of LSCs. Unfortunately, conventional clinical treatments frequently fail to entirely eliminate this key cell population, which often leads to disease relapse and poor survival outcome for patients. In addition, LSCs seem resistant to elimination by cells of the endogenous immune system. The mechanisms are still unknown. Moreover, the use of immune modulating agents, which show promising results in various types of solid tumors, are not efficacious in myeloid neoplasms. Aims: Due the limited knowledge of the interaction between AML LSCs and cells of the adaptive immune system, our major goal is to study the role of T cells in the regulation of LSCs in AML with main focus on CD4+ T cells. Methods: LSCs and CD4+ T cells were isolated from BM and blood of newly diagnosed AML patients. CD4+ T cell states in the BM of AML patients and healthy donors were analyzed by single cell RNA-sequencing. The interaction between T cells and LSCs was studied by co-culture assays followed by functional assays assessing LSCs and CD4+ T cell function such as colony forming and replating assays, flow cytometry and Fluorospot assays. Bulk RNA-sequencing was performed to understand how CD4+ T cells affect LSCs on a transcriptional level. Results: Co-culture of LSCs with autologous BM CD4+ T cells reduced their colony forming capacity. In contrast, LSC function was unaffected when cultured with allogenic AML CD4+ T cells. Similarly, there was no effect on clonogenicity of HSCs in co-culture experiments with autologous CD4+ T cells. Single cell RNAsequencing analysis revealed a cluster of CD4+ T cells that expresses a variety of genes associated with cytotoxicity such as Granzymes and Perforin. We were able to confirm this finding via flow cytometry and could detect a population of Granzyme B expressing CD4+ T cells which has a unique expression pattern of surface markers, for instance, KLRG1+, CCR7- and CD27-. This population was enriched in the BM of AML patients compared to healthy controls. Subsequently, we investigated T cell function via Fluorospot assay. The results show that CD4+ T cells are able to produce and release Granzyme B and IFNγ when co-cultured with LSCs. Blockade of Granzyme B partially restored colony formation in coculture experiments, suggesting that cytotoxic killing is just one mechanism how CD4+ T cells affect LSCs. To determine additional mechanisms, we performed bulk RNA-sequencing of LSCs cultured in presence and absence of autologous CD4+ T cells. RNA-sequencing indicated that CD4+ T cells trigger signaling cascades in LSCs involved in cell cycle stimulation and differentiation, such as upregulation of E2F and MYC target genes and MTORC1 signaling. Summary/Conclusion: Overall, our data shows that CD4+ T cells reduce LSCs in AML by various mechanisms, including direct killing and induction of cell cycle or differentiation. Subsequent experiments will reveal the exact signaling cascades that are triggered in LSCs after interaction with CD4+ T cells. Keywords: CD4+ T cells, Tumor immunology, Acute myeloid leukemia